Metastasis is responsible for most cancer-related deaths and occurs when cancer cells are shed from the primary tumor into the circulation and transported to distant organs. Characterization of circulating tumor cells (CTCs) holds the key to understanding cancer metastasis. Significant progress has been made in recent years in the isolation of CTCs directly from patient blood. Using the latest generation microfluidic technology to capture CTCs, clusters of tumor cells that appear to travel together in the blood stream were unexpectedly captured alongside solitary CTCs. At present little is known about these mysterious circulating cell clusters, but some evidence point to their role in seeding and establishment of secondary tumors. In this P41 Competitive Revision, we proposed to develop new optical technology to meet the needs of biological collaborators who will lead a newly identified driving biomedical project (DBP). The DBP team seeks to determine the origin and in vivo behavior of CTC clusters using animal models of metastasis. Specific, we want to know if multicellular clusters are shed from the primary tumor or if they originate from shed single cells that later grow into clusters. If clusters are pre-formed at the primary site, what is their size distribution and how well do they survive in the peripheral circulation? Are cluster sizes correlated with metastatic capacity? Are host stromal or immune cells involved in the cluster formation? To help answer these questions, we propose to develop in vivo imaging technology that can detect and enumerate CTCs in the circulation of live animals without the need to draw blood samples. We will integrate a two-photon in vivo flow cytometer and imaging cytometer with an optical coherence tomography (OCT) system that is optimized for high-speed cross-sectional imaging of blood vessels with single cell resolution. The technology will enable cancer biologists to track CTCs, characterize their size distribution, and measure clearance rate from the circulation. Ultimately the technology will be useful for answering fundamental questions about cancer metastasis, with the long-term goal of identifying therapeutic targets against metastatic tumors.